1. Field of the Invention
The body's immune system responds to a very large number of different foreign substances (antigens) either by producing antibodies directed against the antigen or cells that react specifically with the antigen.
The reaction between an antibody and its corresponding antigen, which can cause clumping or agglutination, forms the basis for numerous tests involving the detection of antigens or antibodies in fluid samples. Agglutination between an antibody and its corresponding antigen depends upon the formation of intercellular bridges which can sustain detectable agglutination.
Many cells have a small number of antibody combining sites on their surfaces, or the antibody combining sites have a low accessibility to antibodies, with the result that agglutination reactions between the antigens and antibodies do not readily occur. Such antigens are described as "weakly" expressed cell membrane antigens.
Examples of weakly expressed antigens include blood group antigens such as Rh ("D"), Lutheran (Lu), Kell (K), Duffy (Fy), Kidd (Jk), Diego (Di), Yt, Xg and Dombrock (Do). Most of these antigens were discovered by finding immume antibodies either in the mothers of infants with hemolytic disease or in patients who had received multiple transfusions.
Because of the deleterious effects of agglutination, it is desirable to detect weakly expressed antigens. Blood typing (grouping), cross-matching and antibody screening data are needed for safe transfusion and transplantation therapy, management of pregnancy, and evaluation of the newborn.
Other weakly expressed cell membrane antigens are present on other cells such as leucocytes and platelets. Leucocytes and platelets carry many antigens which usually cannot be demonstrated on red blood cells, but which are responsible for the rejection and elimination of foreign tissues. These antigens are collectively termed major histocompatibility complex, or human leucocyte antigens (HLA). The importance of HLA complex matching in renal and bone marrow transplantation is now well established, as is the need for HLA-compatible platelets or granulocytes (leucocytes) for transfusion into highly immunized patients.
Various media and techniques have been developed to promote agglutination for the purpose of detection of weakly expressed antigens. In the area of blood group antigens, one method involves postulated reduction of electrical conductance with high concentrations of hydrophilic colloid, e.g., bovine albumin. With enough antibodies, these tests are quite effective and can be used for Rh blood typing. However, such tests are not very sensitive and not of great value for detecting "weak" Rh and other alloantibodies or antigens. A second method involves breaking the disulfide bonds of the antibody and keeping the bonds from reforming by alkylating the disulfide bonds. Another method involves the use of a low ionic strength saline solution ("LISS"), e.g., glycine with a small amount of sodium chloride or other sodium salt, is added to human serum and cell mixture, and incubated at 37.degree. C. for 15 minutes. Saline washing the addition of antihuman globulin reagent (AHG) or "Coombs" reagent is then carried out.
Several techniques have been developed to identify serum-defined HLA antigens. These tests include leucoagglutination, and microcytotoxicity.
The microcyctotoxicity test involves testing the permeability of cells after incubation with antibody and complement. If antibody is present, it combines with the target cells, as a consequence complement is fixed, and cell permeability increases, which kills the cell. The cell permeability is assessed by adding a solution of trypan blue or eosin which penetrates into dead cells, but leaves viable cells unstained.
Each of these methods involving detection of weakly expressed cell membrane antigens suffers the disadvantage of involving somewhat complicated test procedures. There is a need for a quick and simple method of increasing the sensitivity of detecting weakly expressed cell membrane antigens, including blood type leucocytes and platelets, and antibodies thereto.
2. Description of the Prior Art
Proc. Natl. Acad. Sci., USA, 76(9):4438 (1979) described the enrichment or depletion of membrane cholesterol by contacting the membrane with a media of lipid-modified serum. One composition used was finely dispersed synthetic cholesterol or egg lecithin in human serum medium mixtures; another mixture involved adding synthetic cholesterol or egg lecithin (in tetrahydrofuran or dimethylsulfoxide) to human serum. The authors found that as the cholesterol/phospholipid (C/P) ratio of the mixture was increased to greater than 1, the antigenic expression ("availability") of D antigens increased; conversely as the C/P ratio decreased to less than 1, the antigenic expression decreased. In order to achieve enrichment and subsequent enhancement of antigenic expression, contact of the media for a period of 10 hours at 37.degree. C. was required.
Biochem. and Biophys. Res. Comm., 95(2):887 (1980) described the modification of the cholesterol content of Rh-positive and Rh-negative red cells and quantitated the D binding sites. The authors used the same synthetic cholesterol and egg lecithin dispersions described above and confirmed the above work, i.e., cholesterol enrichment (increase in C/P ratio) enhanced expression of D antigens.
Neither of these articles suggest or describe the detection of weakly expressed cell membrane antigens by use of a lipoprotein substance containing cholesterol and phospholipid, obtained from animal plasma. They also demonstrated a criticality of C/P ratio&gt;1.0 in order to enhance antigenic expression.